PROJECT SUMMARY Revertant mosaicism (RM) occurs from spontaneous, somatic correction of pathogenic mutation, giving rise to areas of normal tissue. Ichthyosis with confetti (IWC), an autosomal dominant skin disorder caused by mutations affecting the tail domains of keratin 10 (K10) and keratin 1 (K1), is remarkable for its high frequency of RM, as patients develop hundreds to thousands of revertant islands of normal skin beginning in childhood. Interestingly, each revertant clone arises from independent copy-neutral loss-of-heterozygosity (CN-LOH), likely via homologous recombination (HR) of the mutant haplotype. Furthermore, the revertant macules are observed to grow in size and number over time, suggesting intercellular competition favoring the selection and expansion of revertant clones over their mutant neighbors. We have successfully demonstrated that expression of IWC mutant keratins uniquely increase the rate of HR, while inducing the formation of DNA double- strand breaks (DSBs). Furthermore, we have developed a conditional knock-in model of IWC, which clinically and histologically recapitulates disease including revertant mosaicism via CN-LOH, and demonstrates expansion of revertant, wild type clones. We now propose to systematically identify and interrogate the mechanisms and mediators governing IWC keratin-induced HR and cellular competition. We will investigate which DNA damage response (DDR) components are recruited to sites of keratin-induced DSBs, characterize the kinetics of DSB formation and resolution, and pinpoint the stage in the cell cycle at which damage occurs. We propose to utilize intravital live-imaging to explore, at the cellular level, whether altered rates of mitosis, apoptosis, or differentiation/delamination underlie the intercellular competition in IWC. Finally, we will perform single-cell RNA sequencing and CRISPR knockout screens, to identify mediators of intercellular competition and determinants or modifiers of HR in IWC. We will further examine compelling candidates in IWC patient and murine tissue and cells. Elucidating the previse mechanisms of genetic reversion and intercellular competition in IWC has the potential to identify pathways which may enable therapeutic recombination to treat inherited and acquired dominant genetic disorders.